the association of hla-dq7 with bullous pemphigoid is restricted to men

FAST-TRACK PAPER

The association of HLA-DQ7 with bullous pemphigoid isrestricted to men

C.C.BANFIELD, F.WOJNAROWSKA, J.ALLEN, S.GEORGE, V.A.VENNING ANDK.I.WELSH*Department of Dermatology and *Oxford Transplant Centre, Churchill Hospital, Headington, Oxford OX3 7LJ, U.K.

Accepted for publication 12 March 1998

Summary This study examines in detail the HLA associations of 74 patients (40 women and 34 men) withbullous pemphigoid (BP) and compares their immunogenetic profile with that of 604 unrelatedcontrol subjects (238 women and 366 men). Correlations were sought between HLA antigens andthe various BP disease parameters investigated. The presence of milia was the only clinical orlaboratory finding which was linked with a specific HLA antigen, HLA-DQ6, in both men and womenwith BP (P< 0·01). BP has previously been linked with the HLA-DQ7 antigen and this associationwas confirmed in 39 of our patients (14 women and 25 men). Twelve of these patients (four womenand eight men) were homozygous for HLA-DQ7. The association of HLA-DQ7 with BP was gender-restricted and only significant for men (P< 0·01). No equivalent HLA disease susceptibility risk factorcould be identified for our female BP patients. This difference in HLA association between men andwomen with BP has not been reported previously, and its significance for disease pathogenesis is notknown. No specific link could be found between HLA-DQ7 and BP for any of the clinical,immunofluorescence, western blotting, treatment or prognostic disease factors studied.

Bullous pemphigoid (BP) is a blistering disease whichprimarily affects the elderly, runs a self-limiting courseand is characterized histologically by subepidermalblister formation. It has been shown to be an auto-immune disease with circulating autoantibodies direc-ted against hemidesmosome antigens1,2 which havebeen demonstrated, in a neonatal mouse model, to bepathogenic.3 Despite individual case reports, however,there is no overall association of BP with any otherautoimmune disease.4 The production of humoral orcell-mediated immunity requires the assistance ofCD4 þ T helper cells. T helper cells are normally onlyactivated by antigens presented in association withclass II major histocompatibility complex (MHC) mole-cules. These are encoded by the genes of the HLA-Dregion on the short arm of chromosome 6. They arenormally expressed on the surfaces of B lymphocytesand antigen-presenting cells of the immune system.The immune response of an individual to a givenpeptide is therefore partly determined by the structure

of the molecules encoded by the HLA-D region of thegenome. The HLA-D region itself is subdivided intothree main genetic subregions termed DR, DP and DQ.Many autoimmune disorders have been shown to havespecific HLA-D associations.5,6

BP has previously been found by ourselves and othersnot to have any HLA class I or II DR associations.4,7–9

Patients with ocular cicatricial pemphigoid (CP), arelated subepidermal blistering disorder, were shown tohave an association with HLA-DQ3. This association wasthen further subtyped by restriction fragment lengthpolymorphisms to the HLA-DQ7 (DQB1*0301) allele8

and this has since been confirmed by polymerase chainreaction sequence-specific probe hybridization experi-ments.10 BP has been found in a small study of 21patients also to share the same HLA-DQ7(DQB1*0301) susceptibility marker.11 Interestingly,many of these earlier immunogenetic studies do notstate the gender of their pemphigoid patients, and nonehas previously reported a gender difference. Our studyexamined the HLA associations of BP and sought torelate them to the clinical, laboratory, treatment andprognostic characteristics of patients.

British Journal of Dermatology 1998; 138: 1085–1090.

1085q 1998 British Association of Dermatologists

Correspondence: Dr C.C.Banfield, Department of Dermatology, StokeMandeville Hospital, Aylesbury, Buckinghamshire HP21 8AL, U.K.

1086 C.C.BANFIELD et al.

q 1998 British Association of Dermatologists, British Journal of Dermatology, 138, 1085–1090

Table 1 HLA antigens in 74 patients with bullous pemphigoid

Sex A A B B C C DR DR DR DR DQ DQ

1 F 11 31 52 60 3 11 13 52 7 62 F 1 2 8 62 3 7 17 11 52 7 23 F 1 7 8 7 17 13 52 2 64 F 11 24 44 45 5 6 17 10 52 2 55 F 2 7 27 1 7 1 9 53 56 F 2 3 7 13 6 7 7 13 52 53 2 67 F 2 24 35 44 4 4 11 52 53 78 F 2 3 7 44 5 7 15 4 53 51 69 F 2 8 60 3 13 52 7 6

10 F 2 11 61 22 7 15 51 53 2 611 F 1 11 52 62 10 15 4 53 51 8 612 F 2 24 62 40 3 15 4 53 51 6 813 F 2 26 27 62 4 10 17 4 52 53 2 814 F 2 28 7 51 7 15 13 52 51 615 F 1 2 44 55 3 5 4 13 52 53 7 616 F 2 31 51 39 1 1 11 52 7 517 F 1 30 7 18 5 7 15 13 52 51 618 F 1 7 8 7 17 15 52 51 2 619 F 23 24 27 14 8 1 8 52 520 F 3 7 18 7 11 14 52 721 F 26 27 38 1 1 13 52 5 622 F 3 31 51 13 6 4 7 53 7 923 F 2 28 8 58 7 17 4 52 53 8 224 F 1 3 8 38 7 17 8 52 225 F 1 11 7 27 1 7 15 11 52 51 7 626 F 2 26 62 39 3 4 12 52 53 7 227 F 2 28 8 57 6 7 17 7 52 53 9 228 F 3 28 7 60 3 7 15 4 53 51 7 629 F 2 33 14 44 5 11 52 730 F 2 28 35 62 4 10 1 4 53 7 531 F 1 2 52 38 15 51 632 M 2 23 35 44 3 1 4 53 7 533 M 2 24 44 62 5 9 4 13 52 53 6 734 M 3 29 44 49 7 11 52 53 2 735 M 2 39 53 4 12 52 736 M 2 3 7 27 1 7 15 51 637 M 2 3 41 70 7 4 13 52 53 7 638 M 1 8 44 5 7 17 4 52 53 2 739 M 1 2 57 56 1 6 15 4 53 51 7 640 M 3 29 62 57 3 6 17 11 52 7 241 M 3 26 35 49 4 7 1 4 53 7 542 M 1 2 37 60 3 6 4 13 52 53 7 643 M 3 24 62 18 3 11 52 744 M 1 8 55 3 17 11 52 7 245 M 11 24 18 55 3 4 11 52 53 746 M 1 8 7 17 9 52 53 2 947 M 29 32 44 5 4 12 52 53 748 M 3 30 62 9 13 52 649 M 24 31 35 40 3 4 4 53 750 M 2 3 44 27 5 7 1 11 52 751 M 24 29 7 44 7 7 8 52 53 2 452 M 3 51 47 6 11 52 753 M 2 3 8 35 4 7 17 52 2 754 M 2 31 35 44 4 5 7 13 52 53 7 255 M 2 60 39 10 7 8 11 3*0202/3 4 756 M 1 29 27 44 2 7 11 52 53 757 M 2 3 7 8 7 15 17 52 51 2 658 M 3 33 7 14 7 8 1 7 5

Subjects and methods

Patients

Seventy-four British caucasian patients (40 women and34 men) who attended a specialized blister clinic in ourdepartment between 1982 and 1995, all with typicalclinical and histological features of BP, positive directimmunofluorescence (IMF) and/or circulating autoan-tibodies that bound epidermally to salt-split skin, wererandomly recruited for our study. Details of the clinicalpresentation and associated autoimmune or malignantdisorders, as well as data on treatment and prognosis,were known for 64 (36 women and 28 men) of thesepatients from a study of their clinical case records.Western blotting was performed for 34 (19 womenand 15 men) of the patients.

HLA typing

Typing of HLA class I and II antigens was performed for73 of our BP patients, on 20 mL of the patients0 wholeblood, at the laboratory of the renal transplant unit,Churchill Hospital, Oxford, using either standard sero-logical or DNA-based techniques which have previouslybeen described in detail.12,13 A group of 604 (238women and 366 men) serologically- and molecularly-typed, random, unrelated, British caucasian organdonors acted as our control population.14

Analysis

Statistical analysis, aided by the Knowledge Seeker

software program (Angoss Software Company Ltd),was performed using the x2 test with Yates correlation.The P-values were corrected to take into account thenumber of antigens tested. Analyses compared theoccurrence of HLA antigens in the BP patient groupwith that in the control population and examined forpossible associations between specific HLA antigens andthe recorded clinical or laboratory findings.

Results

Clinical features and associated disorders

The age of our BP patients at presentation ranged from45 to 93 years with a mean of 74 years. Oral mucosalinvolvement was seen in 18 patients (13 women andfive men) and the presence of milia was noted in 23patients (12 women and 11 men). Seven patients (fivewomen and two men) had an associated internal malig-nancy: three (two women and one man) had bowelcancer, two women had breast cancer, one man hadchronic myeloid leukaemia and one woman had lungcancer. Two patients (one woman and one man) haddiabetes, two (one woman and one man) had perniciousanaemia, two (one woman and one man) had alopeciaareata and one man had autoimmune thyroid disease.

Treatment

All patients were treated with topical clobetasol propio-nate 0·05%. Forty-nine patients (25 women and 24men) required oral prednisolone treatment during the

HLA-DQ7 AND PEMPHIGOID IN MEN 1087


Table 1 (Continued)

Sex A A B B C C DR DR DR DR DQ DQ

59 F 1 62 53 4 960 F 2 11 44 51 5 15 4 7 53 2 861 F 3 24 62 63 9 701 17 1301 3*0101 3*0202/3 2 662 F 1 2 8 39 701 702 8 17 3*0101 2 463 M 2 44 51 5 14 8 11 52 4 764 F 2 24 51 60 1 304 1 12 3*0202/3 5 765 M 1 1401 3*0202 566 F 4 1301 3*0202 53 6 867 M 2 61 62 2 303 1 11 3*0202/3 5 768 M 1 8 701 17 3*0101 269 F 1 27 55 2 6 1 1401/7 3*0202/3 570 M 11 15 51 52 6 771 F 2 24 60 62 9 10 1302 52 672 F 1 24 8 60 7 10 4 17 52 53 2 873 M 1 3 44 56 1 5 52 674 M 2 11 27 2 7 17 3*0202 53 2

course of their disease, 32 (20 women and 12 men)needed sulphonamide treatment, 17 (10 women andseven men) were treated with azathioprine, 14 (sixwomen and eight men) had oral minocycline therapyand two (one woman and one man) receivednicotinamide. One woman developed symptomaticosteoporosis after commencing oral prednisolonetreatment.

Disease course and severity

Thirty-one patients (18 women and 13 men) wereadmitted for in-patient treatment, a crude marker ofsevere disease, and three (one woman and two men)were admitted on more than one occasion. The meanduration of an in-patient admission was 23 days. Clin-ical remission was noted in 42 patients (24 women and18 men) during the study period and the averageduration to remission was 22 months.

Immunofluorescence

Direct IMF was positive for IgG in 40 patients (20women and 20 men), for C3 complement in 53 (29women and 24 men) and for IgA in four (two womenand two men). Indirect IMF with intact skin was positivefor IgG in 54 patients (30 women and 24 men), for C3in six (three women and three men) and for IgA in onlyone man. Indirect IMF performed with 1 mol/L NaClsplit skin was positive for IgG in 55 patients (32 womenand 23 men), for C3 in eight (four women and fourmen) and for IgA in six (four women and two men).

Western blotting

Western blotting, using the patients0 sera, showed thatsera from 21 patients (14 women and seven men)bound the 230 kDa BP antigen 1 (BPAG 1), sera fromseven (three women and four men) bound the 180 kDa

BP antigen 2 (BPAG 2) and sera from five (two womenand three men) bound both BPAG 1 and BPAG 2. Serumfrom one man bound a 285 kDa basement membranezone (BMZ) antigen and serum from another manbound a 250 kDa antigen. Sera from nine patients(four women and five men) were negative on westernblotting.

HLA class I and class II antigens

No significant associations were detected between theproducts of the HLA-A, B or C genetic loci and our BPpatients (Table 1), nor was any significant associationfound between our BP patients and the HLA-DR class IIantigens. Results of the HLA class II DQ typing in our BPpatients compared with the control population areshown in Table 2. HLA-DQ7 was associated with BPin 39 patients (14 women and 25 men; P< 0·01).Twelve of the patients (four women and eight men)were homozygous for HLA-DQ7 in comparison with 28out of 604 control subjects (P< 0·01). Intriguingly,analysis of this association showed that the associationof HLA-DQ7 was only significant for our male BPpatients (P< 0·01). Overall, 25 of the men with BP(74%) had HLA-DQ7 and in contrast only 31% of ourcontrol population had this particular antigen. Four-teen of our female BP patients (36%) had the HLA-DQ7antigen and four were homozygous for HLA-DQ7, butcomparison with our control population showed thatthis finding was not significant. HLA-DQ7 was notlinked with any of the clinical or treatment parametersstudied and it did not appear to affect disease prognosis.There was also no association between HLA-DQ7 andresults of the IMF investigations, nor with the targetantigens detected by western blotting.

The HLA-DQ8 antigen was present in seven womenwith BP and in none of the men with BP (P< 0·01),but HLA-DQ8 was not associated with an increased riskof BP. We have analysed and found no significant



HLA-DQ Female BP patients Male BP patients Control subjectsantigen n ¼ 39 (%) n ¼ 34 (%) n ¼ 604: 238 F, 366 M (%)

DQ2 15 (38) 11 (32) 257 (43)DQ4 1 (3) 3 (9) 25 (4)DQ5 8 (21) 5 (15) 176 (29)DQ6 19 (49) 9 (26) 249 (41)DQ7 14 (36) 25 (74)* 189 (31)DQ8 7 (18) 0 (0) 111 (18)DQ9 2 (5) 1 (3) 60 (10)

* P<0·01

Table 2 HLA-DQ antigens in bullouspemphigoid (BP) patients and controls

differences in HLA-DQ7 and HLA-DQ8 antigensbetween the sexes for our control population. In ourstudy, the only clinical feature or laboratory finding thatwas associated with a specific HLA antigen was thepresence of milia which was linked with the HLA-DQ6antigen in 13 (seven women and six men) of the 23 BPpatients who had this particular clinical sign (P< 0·01).

Discussion

BP and CP are closely related subepidermal immuno-bullous disorders which share many clinical features,immunopathological findings and also BMZ target anti-gens. BP and CP have previously been shown not to belinked with any HLA class I or II DR antigens,7–9 butboth disorders have been found to be associated with theHLA-DQ7 antigen.8,10,11,15 None of the previous immu-nogenetic studies has reported a gender difference.There has also been some speculation that the HLA-DQ7 antigen is important either in the elicitation of anautoantibody response against the target antigens of theBMZ,11 or that it influences the clinical phenotype of theblistering disease, e.g. by predisposing specifically tomucosal disease and the development of ocular scarringin patients with CP.15 Our study confirms that the HLA-DQ7 antigen is significantly associated (P< 0·01) with asusceptibility to BP, but surprisingly only in men.Although the HLA-DQ7 antigen determines diseasesusceptibility it does not seem to influence the clinicalfeatures, the detection of autoantibodies, the treatmentor the prognosis of our male BP patients who have thisparticular antigen. Interestingly, the HLA-DQ8 antigenwas only found in women with BP, but it was notassociated with an increased risk of the developmentof BP, and no comparable immunogenetic risk factorcould be identified in our female BP patients. In ourstudy, the autoantibodies which were detected andbelieved to be implicated in the causation of BP do notseem to be linked directly with any HLA immunogeneticrisk determinant, and the exact role antibody-mediatedinjury plays in the pathogenesis of BP remains unclear.Ocular scarring and oral ulceration are the cardinalclinical features of CP. In contrast, oral mucosae invol-vement is only seen in a minority of BP patients and ourstudy did not find any evidence to support the sugges-tion that the HLA-DQ7 antigen predisposes towardsmucosal disease in patients with BP. The association ofBP in men with a specific HLA class II, immunogeneticdeterminant provides further support for the conceptthat BP is an autoimmune disease. However, the factremains that a considerable proportion of pemphigoid

patients do not have the DQ7 allele.10,11,15 The HLA-DQ7 antigen therefore appears to exert a subtle and asyet unknown influence on the development of thepemphigoid group of bullous diseases.

An earlier study of 38 BP patients has suggested thatHLA-B7 is associated with a poor response to immuno-suppressive therapy.9 This association was not found byour study and neither the treatment nor the prognosisof our group of BP patients appears to be linked withany specific HLA risk factor. Milia are small, superficialkeratin retention cysts which typically arise in the skinof patients injured by the subepidermal blistering pro-cess. We found that for our BP patients, milia formationwas significantly associated with the HLA-DQ6 antigen(P< 0·01). The explanation for this association is atpresent unknown: perhaps the HLA-DQ6 antigen influ-ences either keratinization or the epidermal tissue repairresponse.

In conclusion, our study, the largest so far, confirmsthat BP is associated with the HLA-DQ7 antigen butinterestingly only in men. This unexpected finding is allthe more intriguing since it does not appear to beassociated with any other clinical, laboratory or treat-ment parameter of BP and neither does it seem toinfluence the prognosis of this disease. Further studiesare required in order to investigate the intriguing andincreasingly complex role of HLA antigens in the patho-genesis of the pemphigoid group of subepidermalblistering disorders.

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major BP antigen. J Invest Dermatol 1989; 92: 33–8.2 Giudice GJ, Squiquera HL, Elias PM et al. Identification of two

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4 Taylor G, Venning VA, Wojnarowska FT et al. Bullous pemphigoidand autoimmunity. J Am Acad Dermatol 1993; 29: 181–4.

5 Tussey LG, McMichael AJ. General introduction to the MHC. In:Modulation of the MHC Antigen Expression and Disease (Blair GE,Pringle CR, Maudsley DJ, eds ). Cambridge: Cambridge UniversityPress, 1995; 1–25.

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11 Delgado JC, Turbay D, Yunis EJ et al. A common major histocom-patibility complex class II allele HLA-DQB*0301 is present inclinical variants of pemphigoid. Proc Natl Acad Sci USA 1996;93: 8569–71.

12 Marren P, Yell J, Charnock FM et al. The association betweenlichen sclerosus and antigens of the HLA system. Br J Dermatol1995; 132: 197–203.

13 Bunce M, O’Neill CM, Barnardo MCNM et al. Phototyping: com-prehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4,DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequencespecific primers (PCR-SSP). Tissue Antigens 1995; 46: 355–67.

14 Bunce M, Barnardo MCMW, Procter J et al. High resolutionHLA-C typing by PCR-SSP. Identification of allelic frequenciesand linkage disequilibria in 604 unrelated random UK cauca-sians and a comparison with serology. Tissue Antigens 1996; 48:680–91.

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the association of hla-dq7 with bullous pemphigoid is restricted to men

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